The coat protein clathrin is required for localization of resident proteins to the Golgi, for proper sorting of proteins to the lysosome, and for receptor-mediated endocytosis. At present, only a few proteins required for Golgi protein localization have been identified, and the mechanism by which these proteins act with clathrin to localize resident Golgi proteins is incompletely understood. Therefore, this proposal focuses on the isolation and characterization of additional components of the Golgi localization machinery in the organism, Saccharomyces cerevisiae. Clathrin-interacting proteins will be identified in a genetic screen for mutations that cause a synthetic growth defect in cells expressing a functionally-compromised clathrin heavy chain encoded by the chcl-ts allele. New mutants (denoted sct for synthetic lethal with chcl- ts) from a preliminary screen have been classified using three biological assays to identify those mutations that specifically affect Golgi protein localization. The protein localization defect in this class of sct mutants will be characterized by assaying for the mislocalization of two resident proteins each from the trans and medial Golgi compartments, and one from the endoplasmic reticulum. Two mutants with strong effects on Golgi protein localization will be analyzed more extensively. First, the wild type copy of each gene will be cloned, sequenced, and examined for homology to known proteins. Second, immunofluorescence and density gradient centrifugation and fractionation, will be used to localize each protein to a subcellular compartment. Finally, native immunoprecipitations and cross-linking experiments will be used to detect direct interactions between the sct proteins and other proteins required for Golgi protein localization.